Sulfite waste liquor can be employed for the recovery o f proteins from dilute solutions. A m ethod is described for wheat m ashes. For m axim u m recovery, protein hydrolysis should be avoided, and the acidity o f the m ash should be adjusted to a pH range o f 1.5 -2 .5. Two parts o f the sulfite waste liquor, calculated on the basis o f its lignin content, are required to recover 5 parts o f protein. During or after
lignin addition, the m ash is heated to at least 85° C. and is preferably cooled before filtration. Under these conditions 85 to 9 0 % o f the total m ash protein (or 80 to 8 5 % o f the solu
ble protein) is readily recovered, and the liltcrability is markedly improved when compared with that o f untreated m ashes. The recovered protein shows prom ise as a feed, when properly supplem ented.
James S. Wallerstein, Eduard Färber,
Gertrude D. Maengwyn-Davies, and Arthur L. Schade
O V E R L Y B IO C H E M IC A L R E S E A R C H FO U N D A TIO N , IN C ., NEW Y O R K 1, N. Y .
W
HEN cereals are used as the carbohydrate source in industrial fermentations, the protein content of the grain represents a valuable by-product. In view of the shortage of protein under wartime conditions, its recovery is important for the national economy. Furthermore, by such recovery the amount of wastes from fermentation industries will be reduced and stream pollution diminished. Various methods for protein recovery, such as screening, spraydrying of slops, the sulfite process (I), etc., are in use. A new method for protein recovery by precipitation with sulfite waste liquor is described here as applied to wheat mashes; it may be used with equal success for other cereals. The advantages are a high recovery of protein, low material and handling costs, and the use of conven
tional equipment.
The ability of some constituents of sulfite waste liquor to com
bine with protein is made use of in the tanning industry. It has been shown that the lignosulfonic acid effects a chemical com
bination with hide proteins (3, 7). A study of the depressive effect of lignin upon the decomposition of proteins by bacteria showed that a complex is formed when lignin and gliadin (5, 6), or casein, were allowed to interact. This lignin-protein complex possessed the various chemical, physicochemical, and biological properties characteristic of the major portion of the organic matter (humus) of the soil. Recently, proteins were used as a lignin precipitant in the purification of wood liquors (3).
In 1942 research was-started in this laboratory on the produc
tion of glycerol from wheat by fermentation. Initially, second clears flour, and later, granular wheat flour, were employed. Since sizable quantities of nitrogenous matter interfere with the dis
tillation of fermentation glycerol in various ways, experiments for the removal of proteins with sulfite waste liquor were undertaken for prepurification of the mash prior to fermentation. When the maximum amount of protein was removed by sulfite waste liquor treatment, it was found that distillation difficulties were sub
stantially eliminated, the amounts of residue and solids in the distillations were reduced, and a greater purity of end products was obtained.
Initial experiments indicated that the quantity of protein re
moved by sulfite waste liquor is inversely proportional to the degree of its disaggregation. Therefore a method was employed by which the protein in the mash undergoes as little breakdown as possible prior to precipitation.
M A T E R IA L S AND M E T H O D S
The granular wheat flour used had the following analysis:
moisture 11.0%, protein 10.3%, carbohydrates (calculated as glucose) 77.9%, ash 0.6%. The flour was converted into a fermentation mash with a minimum of proteolysis by the follow
ing method: A premalt was prepared by mixing 50 grams of high-diastatic malt with 500 ml. of water, stirring the mixture at 37° C. for 30 minutes to free the diastases for maximum activity, and then for an additional half hour at 60° C. to inactivate the proteases of the malt. One kilogram of granular wheat flour, suspended in 5 kg. of water, was liquefied by heating to 85° C.
after addition of one fifth of the premalt. After liquefaction of the starch, the mash was cooled to 62° C., and the remaining four
T a b l e I. C o m p a r i s o n o r P r o t e i n - P r e c i p i t a t i n g A c t i o n o f S a m p l e s F a n d M
Lignin Added. Mg.
Sample F Sample M
0 0
0 .7 8 (3 )a 1.50 (6) 2 . 0 ( 10) 5 .2 (20) 7.' 8 (30) 10.4 (40) 2(L8 (80) 3 Ü 2 (120) 4 1 .0 (100)
3 .9 ( 5 .0 7 ) 7.'8 (11.34)
15.' ¿ (2 2 .0 8 ) 3 Î .2 (45.30)
T otal N& in
•"¡Urate of Lignin- % Sol.
Protein %
T otal Sol.
Treated Sample, and Insol. Pro
Mg. Rem oved tein Recovered
0 .3 0 39
5.01 9 .5 44.9
4 .7 2 25.0 5 4.2
4 .0 0 2 0.0 5 4.8
4.17 33.5 5 9.5
3 .4 5 4 5.0 06.0
2 .9 9 5 2.5 7 1 .0
2.39 02.0 7 0.8
1.22 8 0 .5 88.1
1.32 7 9.0 8 7 .2
1.12 8 2 .0 8 9.1
1.23 8 0.5 8 8.1
1.10 8 2 .5 8 9.3
1.07 83 8 9 .6
° Figures in parentheses represent mg. o f samples F and M used.
Calculated after deduction of nitrogen | and M added (F - 0.80, M
•gen present in amounts of samples F 0.70 mg. N per gram).
772
August, 1944 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 773 fifths of the premalt was added with stirring. The mash was
then malted for 2 hours at 62° C. and for an additional 2 hours at 52° C. The malting time is not critical and may be appreciably shortened. Instead of malt, other amylolytic materials, such as bacterial amylase, can be employed.
Two samples (F and M ) of lime-neutralized, concentrated
Ash (as residue after ignition) CaO
The actual precipitating agent was lignosulfonic acid, expressed in terms of lignin in accordance with the common analytical content after conversion into free lignosulfonic acid (a procedure suggested by the manufacturer).
T o 22.675 grams of oven-dry sample M suspended in 100 ml. of water, 1.22 ml. (2.243 grams) of concentrated sulfuric acid (95.5%), diluted to 8 normal strength, were added under stirring.
The mixture was heated to 85° C. and filtered, the precipitate was washed, and the combined filtrate and washings were made up to 200 ml. with distilled water. One milliliter of this stock solu
tion corresponds to 0.078 gram of lignin, calculated on the basis of the manufacturer’s analysis.
For a more general comparison of the protein-precipitating action of the two samples of sulfite waste liquor, sulfosalicylic acid was employed; as stock solution, 1 0 grams of this acid (reagent grade) were dissolved in distilled water and brought to a volume of 100 ml. (pH = 1). The semimicro-Kjeldahl method was used for the nitrogen analyses. The factor 6.25 was taken for the conversion of nitrogen into protein. The pH determina
tions were made with a Beckman meter.
Experiments were undertaken to determine the optimum condi
tions for the precipitation of proteins in the mash. When the malted wheat flour mash was acidified to pH 2.0, approximately the optimal pH for precipitation with sulfite waste liquor, 39%
of the protein originally present was insoluble and could be
re-moved by filtration with Celite 512. The action of sulfite waste liquor was studied on the soluble 61 % of the protein.
V A R IA T IO N IN Q U A N T IT Y O F PR O TE IN P R E C IP IT A N T S The malted mash was acidified to approximately pH 2.0, diluted to four times its original volume with distilled water, and filtered with Celite 512. Each 100 ml. of the clear filtrate used for the following experiments contained an equivalent of 6.25 grams of granular wheat flour (392 mg. of soluble protein).
One milliliter of solution, containing different amounts o f samples F and M by appropriate dilution of their stock solutions, was added to 10 ml.»of the filtrate. The mixture was heated to boiling, readjusted to the preboil weight with distilled water, and filtered with Celite 512. One milliliter of distilled water was
The protein-precipitating action of the sulfite waste liquor sam
ples was compared with that of sulfosalicylic acid. Five milliliters of varying concentrations of sulfosalicylic acid made by dilution of the stock solution with 0.1 N hydrochloric acid were added to 10 ml. of filtrate; the mixtures were left in the incubator for 40 minutes (4) and then filtered with Celite 512. As control, 5 ml.
of 0.1 N hydrochloric acid were added to 10 ml. of filtrate, and the mixture was left in the incubator for 40 minutes.
SUIF0SAUCYUC ACID MGS.
Figure I. Comparison o f Sulfosalicylic Acid and 2 Sulfite W aste Liquors (on Basis o f Lignin Content) as Protein Précipitants
The percentage of soluble nitrogen removed from the mash filtrates with increasing amounts of sulfosalicylic acid and of the sulfite waste liquor samples in terms of-their lignin contents are shown in Figure 1. These curves, which are representative of the experiments, indicate that sulfite waste liquor is a better precipi
tant for wheat proteins than sulfosalicylic acid; in order to achieve the same degree of precipitation, more than ten times as much sulfosalicylic acid was needed than lignin, and the maxi
mal percentage of protein precipitated was considerably higher with the latter agent.
774 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 36, No. 8 V A R IA T IO N IN pH
The effect of pH variation on the precipitation of protein was determined with the optimum addition of sample F. Samples of the mash used in the precipitation experiments were adjusted by- addition of sulfuric acid or sodium hydroxide to the desired pH, made up to a final dilution of 1 to 4, and filtered with Celite 512.
The pH values of the filtrates are shown in Figure 2. Ten milli
liters of these filtrates were treated with 1 ml. of sample F con
taining 15.6 mg. of lignin. One milliliter of distilled water was added to 1 0 ml. of the filtrate in each case and used as control.
The treated samples and controls were heated to boiling, cooled, brought to original weight with distilled watftr, and filtered with Celite 512.
Figure 2. Influence o f pH on Precipitation o f Protein by Sulfite W aste Liquor
Figure 2 compares the nitrogen contents of the treated samples and controls at varying pH values. The upper curve (controls) indicates that the amount of soluble nitrogen (in other words, the amount of nitrogenous matter remaining in the filtrates) changes considerably with the pH of the mash; it is at a maximum at pH 2.75. Maximum precipitation of this soluble nitrogenous matter by addition of the optimum amount of sulfite waste liquor is obtained between the pH range of 2 and 4. This fact is derived from an examination of the lower curve (treated samples). The yield of precipitated protein calculated on the basis of the sum of soluble and insoluble nitrogen is greatest in the pH range of 1 to 3. Results of pH studies on sample M closely resemble those for sample F.
D ISCU SSIO N
These studies on the precipitation of wheat proteins show that, with sulfite waste liquor, 80-S5% of the soluble protein in a di
luted mash is readily recovered. For experimental purposes, attention was directed chiefly to the soluble protein of the mash.
However, when fermentation mashes of the usual concentrations containing insoluble as well as soluble protein were treated under optimal conditions for the recovery of soluble proteins, not only
was 85-90% recovery realized, but also the filterability of the fermentation mash was greatly improved. The fermentation was not affected by this treatment.
To recover 85-90% of the protein from a fermentation mash, the acidity was adjusted to a pH value in the range 1.5-2.5.
Sulfite waste liquor in an amount equal to 2 parts of lignin for every 5 parts of protein was added to the adjusted mash. The treated mash was heated to 85-90° C. for 10 minutes with thor
ough mixing. For the highest recovery of protein, it is pref
erable to filter after cooling the mash to room temperature. If desired, the filter cake can be washed free of sugar and the wash water used for subsequent mashings. The protein-lignin cake can readily be converted to a dry form following the usual pre
cautions for drying such materials.
Proteins may be recovered by this method either from mashes prior to fermentation or from fermentation slops, such as slops from alcohol distilleries. If used for alcohol slops, the method not only provides for the recovery of a material high in protein but also leads to a considerable purification and thereby a lowering of the B.O.D. and solids content of the effluent.
Preliminary tests on the feeding of the dried protein-lignin to rats indicated that this material is a satisfactory source of pro
tein, provided the diet is supplemented by the essential amino acids known to be lacking in wheat protein. With such a supple
ment the rats maintained satisfactory growth rate.
While the process described was developed for the recovery of wheat proteins, it may also be applied to proteins from other vegetable sources such as corn and potatoes. Yields for the latter are of the same order as those for wheat.
S U M M A R Y
1. A method is described for the recovery of protein by the addition of sulfite waste liquor to hot, acidified wheat mashes.
By this method 85-90% of the protein is readily recovered and filtration of the mash is greatly improved.
2. Studies with two different sulfite waste liquor samples indi
cate that the protein-precipitating action is a function of lignin (lignosulfonic acid) content. For the precipitation of soluble wheat proteins, the optimum ratio of lignin to protein is 1 to 4.
3. The effectiveness of lignosulfonic acid for the precipitation of soluble wheat protein was compared to that of sulfosalicylic acid. The relative activity of lignosulfonic acid was more than ten times as great as that of sulfosalicylic acid. Further, the maximum amount of precipitation achieved by lignosulfonic acid was considerably higher.
4. The optimal range of acidity for maximum precipitation of soluble protein is pH 2 to 4.
5. With fermentation mashes containing both soluble and insoluble protein, approximately 2 parts of sulfite waste liquor, calculated on the basis of its lignin content, are required to recover 5 parts of protein. The optimum pH range for this precipitation is 1.5 to 2.5.
6. Preliminary tests indicate that the recovered protein is suitable for feed purposes, when properly supplemented.
A C K N O W L E D G M E N T
T h e au th ors are in d eb ted t o A rn old Schein o f the F low er an d F ifth A v en u e H osp itals for th e ra t assays.
L IT E R A T U R E C IT E D
(1) Balls, A . K ., Hale, W . S., R ose, W . G ., T ucker, I. W ., Axelrod, B ., Schimmer, S., Peruzzi, J. J „ and W alden, M . K ., Com m un.
from Agr. Research Adm inistration, Sept. 17, 1943.
(2) Farber, Eduard, unpublished com m unication, 1942.
(3) G ustavson, K . H ., Svensk Papperstidn., 44, 193-200 (1942).
(4) M aw son, C . A ., Biochem. J „ 36, 273-80 (1942).
(5) W aksm an, S. A ., and Iyer, K . R . N ., Soil Set., 34, 43 (1932).
(6) Ibid., 34, 71 (1932).
(7) W ilson, J . A ., and P orth, I. H ., J. A m . Leather Chem. Assoc., 38, 20-30 (1943).